The mechanism of removal of the intervening sequence from yeast tRNA precursors is under investigation. We have shown that a soluble extract from yeast contains the splicing activities. In the absence of ATP only the cleavage of the precursor occurs via two endonucleolytic cuts which produce 3'-phosphate and 5'-hydroxyl termini on the intervening sequence and half-tRNA molecules. These half-tRNA molecules can be efficiently ligated in an ATP-dependent reaction. Further investigation of tRNA splicing will follow three major directions. First, the enzyme, or enzymes, involved in tRNA splicing will be characterized. Purification of the two activities should reveal whether the endonuclease and ligase activities are two physically distinct enzymes or part of a single multifunctional enzyme. A detailed study of the purified activities is planned with a view to establishing the mechanism through kinetics, mode of action inhibitors and other physical and chemical techniques. Second, a study of RNA structure will be pursued. Structural analysis will be conducted by means of observing the relative accessibility of various nucleotides to chemical modification or ribonuclease cleavage. In addition synthetic substrates which vary in sequence and/or structure will be utilized to study the structure-function relationship of the ligase and endonuclease substrates with the enzymes. Third, sequencing of the other, as yet unidentified, pre-tRNAs will continue. It is anticipated that additional primary sequence information will expand our knowledge of common structural features among the pre-tRNAs and contribute to the directions taken in producing model substrates and in other structural studies.